1. Field of the Invention
The present invention relates to a novel strain of mycoplasma isolated from a patient with AIDS. The mycoplasma does not appear to be related to any other species of human or animal mycoplasma. This novel mycoplasma is referred to hereinafter as M. penetrans.
The invention also relates to use of the mycoplasma M. penetrans in detecting specific antibodies in sera of patients with AIDS or sub-acute and acute fulminant systemic diseases and/or animals and its use as a vaccine against infection by the mycoplasma. The invention further relates to M. penetrans-specific antibodies and cross-reactive M. penetrans antibodies (i.e. antibodies to homologous antigenic determinants), including monoclonal antibodies of each, which are useful in detecting M. penetrans antigens in infected tissues and specimens of patients or animals. The invention also relates to M. penetrans DNA probes which are useful in detecting M. penetrans genetic materials in infected tissues, blood or body fluids of patients or animals. M. penetrans genetic materials may also be detected in infected humans or animals by using M. penetrans DNA sequences or a homologous M. penetrans DNA sequence and the polymerase chain reaction ("PCR") (U.S. Pat. No. 4,683,202 incorporated herein by reference).
The ability to monitor AIDS or other acute fulminant systemic disease status can be of great value. In addition to improving prognostication, knowledge of the disease status allows the attending physician to select the most appropriate therapy for the individual patient, e.g. highly aggressive or less aggressive therapy regimens. Because of patient distress caused by more aggressive therapy regimens, it is desirable to distinguish those patients requiring such therapies.
Mycoplasma is a genus of cell wall-less sterol-requiring, catalase-negative pathogens commonly found in the respiratory and urogenital tracts of man and other animals. The cells of Mycoplasma are typically non-motile and pleomorphic, ranging from spherical, ovoid or pear-shaped to branched filamentous forms. Filaments are the typical forms in young cultures under optimal conditions, which subsequently transform into chains of coccoid cells which later break up into individual cells that are capable of passing through membrane filters of pore size 0.45 .mu.m or even 0.22 .mu.m.
A trilaminar cytoplasmic membrane contains sterols, phospholipid and proteins. Therefore, the cells are generally susceptible to polyene antibiotics and to lysis by digitonin.
Replication of the Mycoplasma genome may precede cytoplasmic division resulting in multinucleate filaments before individual cells are delimited by constriction. Budding can also occur.
Most Mycoplasma species are facultatively anaerobic, and all known species are chemoorganotrophic. The fermentative species of Mycoplasma utilize sugars such as glucose, while non-fermentative species can hydrolyze arginine. Only a few species of mycoplasma can utilize both glucose and arginine to grow.
Known mycoplasmas may be grown on complex media, such as Hayflick medium, while fastidious mycoplasmas may be grown on diphasic SP-4 medium. The colonies are usually of the "fried egg" type, i.e., an opaque, granular central region, embedded in the agar, surrounded by non-granular surface growth. The optimal growth temperature of mammalian strains is 36.degree.-37.degree. C.
Many species of Mycoplasma produce weak or clear hemolysis which appears to be due to the secretion of H.sub.2 O.sub.2. This H.sub.2 O.sub.2 secretion is believed to be responsible for some aspects of the mycoplasmas' pathogenicity. Known mycoplasmas are commonly sensitive to chloramphenicol and tetracyclines.
The Mycoplasma genus currently consists of more than 60 known species which are differentiated on the basis of various tests, including utilization of glucose and mannose, arginine hydrolysis, phosphatase production, the "film and spots" reaction and haemadsorption.
Mycoplasmas are the smallest and simplest free-living organisms known. Mycoplasmas are not obligatory intracellular microorganisms and are usually found extracellularly, but can be found intracellularly in the infected tissues (Mycoplasma, Eds. Wolfgang, J. J., Willette, H. P., Amos, D. B., Wilfert, C. M., Zinsser Microbiology 19th Ed. 1988, Appleton and Lange, 617-623). The term mycoplasma apparently was first used by B. Frank in 1889 (Frank B., Dent. Bot. Ges., 7, 332 (1889) and Krass, C. J. et al., Int. J. Syst. Bacteriol. 23, 62 (1973)). Frank, after careful microscopic observation, began writing about invasion of plants (legume) by these microorganisms and stated: "the changed character of the protoplasm in the cortical cells arising from infection, I will designate as mycoplasma". Later, he had more explicitly defined mycoplasma as a mixture of small fungus-like microorganisms and cell protoplasm (Frank, B., Landwirt. Jahrb. 19, 523 (1890)). The description reflected the difficulty of differentiating this unique microorganism from the infected host cells morphologically.
Even today with electron microscopy, it is still often difficult to differentiate the mycoplasmas from the cellular protoplasmic processes or the subcellular organelles of the infected host, because ultrastructurally, these microorganisms have protoplasm-like internal structures and are bounded by only an outer limited membrane (unit membrane) without a cell wall. Thus, there have been few electron microscopic studies of mycoplasmas identified directly in the infected tissues of animals or humans.
It has been reported that ultrastructural examination of infected tissues has failed to localize the microbe, even in tissues where very high titers (&gt;10.sup.9 /gm) of microorganisms were recovered in culture (Elizan, T. S. et. al., Pro. Soc. Exp. Biol. Med. 139, 52 (1972) and Schwartz, J. et al., Pro. Soc. Exp. Biol. Med. 139, 56 (1972)). Therefore, morphologically, the microbe might be mimicking certain normal cellular or subcellular structures in the infected host tissues and preventing direct identification.
In addition to the natural difficulty of morphological differentiation between the microorganisms and the protoplasm of infected cells, the often poorly preserved formalin-fixed clinical materials present further limitations to any attempt to directly visualize mycoplasma organisms in the tissues.
Mycoplasmas cause a variety of diseases in animals, plants and insects. However, the roles of known human mycoplasmas except for M. pneumoniae in causing a typical penumonia are difficult to assess. Urines or urogenital swabs from healthy people and patients with a variety of different diseases have already been examined extensively. U. urealyticum and M. hominis were the most commonly found mycoplasmas. Taylor-Robinson, D., et al., Nature 222, 274 (1969); Tarr, P. I., et al., J. Infec. Dis 133, 419 (1976); Taylor-Robinson, D., et. al., N. Eng. J. Med. 302, 1003 (1980); Fiacco, V., et al., J. Clin. Microbiol. 20, 862 (1984). Although a wide variety of urogenital and reproductive disorders have been associated with U. urealyticum and M. hominis, it has been difficult to establish a definite etiological role for them because they are often found as frequently in patients without disease as in those with disease. Taylor-Robinson, D., et al., N. Eng. J. Med. 302, 1003 (1980). M. genitalium was also isolated from two patients with non-gonococcal urethritis. However, most patients with urogenital diseases do not have clear evidence of M. genitalium infection. Tully, J. G., et al., Lancet 1, 1288 (1981). On the other hand, mycoplasmas are known to be extraordinary pathogens, capable of causing chronic debilitating diseases and producing a variety of clinical manifestations in animals and frequently suppressing host immune defense mechanisms. The Mycoplasmas, Vol. IV, Razin, S., Barile, M. F., eds., Academic Press, pp. 203-286 (1983).
Recent studies suggest mycoplasmas may play an important role in the diseases progression of AIDS. Lo, S-C., et al., Am. J. Trop. Med. Hyg. 40, 213 (1989); Lo, S-C., et al., Am J. Trop. Med. Hyg. 41, 601 (1989), Montagneir, L., et al., C.R. Acad. Sci. Paris 311, 425 (1990); Chowdhury, M. I. H., et al., Lancet 336, 247 (1990); Bauer, F. A., et al., Hum. Pathol. 22, 63 (1991); "Mycoplasma and AIDS--what connection?" [editorial], Lancet 337, 20 (1991); Lo, S-C., et al., Science 251, 1074 (1991). For example, Mycoplasma fermentans systematically infects many patients with acquired immunodeficiency syndrome (AIDS), appears to be associated with diseased conditions in various organs and tissues, and was recently associated with development of nephropathy in AIDS patients. Lo, S-C., et al., Am. J. Trop. Med. Hyg. 40, 213 (1989); Lo, S-C., et al., Am J. Trop. Med. Hyg. 41, 601 ( 1989); Bauer, F. A., et al., Hum. Pathol. 22, 63 (1991). Examination of polymerase chain reactions and cultures of urines revealed a high prevalence of M. fermentans infection in AIDS patients but not in non-AIDS controls.
2. Description of the Background Art
Acquired Immune Deficiency Syndrome (AIDS) is a devastating disease that has afflicted over one million people worldwide (AIDS Weekly Surveillance Report--United States, Centers for Disease Control, Aug. 29, 1988). The disease is clinically characterized by a set of typical syndromes which manifests itself by the development of opportunistic infections such as pneumocystic carinii pneumonia (PCP), toxoplasmosis, atypical mycobacteriosis and cytomegalovirus (CMV). Further characteristics of the AIDS associated syndromes are the clinical manifestation of neuropsychiatric abnormalities, of AIDS encephalopathy (Naura, B. A., et al., Ann.Neuro 19, 517 (1986)), kidney failure of AIDS nephropathy, heart failure of AIDS cardiomyopathy infections and certain uncommon malignancies such as Kaposi's sarcoma or B-cell lymphoma (Durack, D. T., N.Eng.J.Med. 305, 1465 (1981); Reichert, C. M., et al., Am.J.Path. 112, 357 (1983); Ziegler, J. L., et al., N.Eng.J.Med. 311, 565 (1984)).
Through co-cultivation of AIDS patients' peripheral blood cells with mitogen-stimulated normal human lymphocytes or permanent human T-cell lines, a number of laboratories have isolated T-cell-tropic human retroviruses (HIV), Barre-Sinoussi, F., et al., Science 220, 868 (1983); Gallo, R. C., et al., Science 224, 500 (1984). Epidemiologically, the newly isolated retroviruses have been shown to be highly associated with patients of AIDS and/or AIDS-related complex (ARC). Schupback, J., et al., Science 224, 503 (1984); Sarngadharan, M. G., et al., Science 224, 506 (1984). In vitro studies with HIV have demonstrated T-cell tropism and cytopathic changes. Barre-Sinoussi, F., et al., supra; Popovic, M., et al., Science 224, 497 (1984). HIV is believed to be the causative agent of AIDS.
However, the establishment of an animal model of AIDS by HTLV-III-LAV injection has not been successful. Gajdusek, D. C., et al., Lancet I, 1415 (1984). The chimpanzee is the only primate other than man found to be susceptible to infection by HIV. However, overt AIDS manifested by the development of opportunistic infections and/or unusual malignancies has not yet been seen, despite evidence for persistent infection and/or viremia in experiments on this species. Gajdusek, D. C., et al. Lancet I, 55 (1985). Thus, the human retroviruses have not fulfilled Koch's postulates, i.e., producing transmissible AIDS-like diseases in experimental animals. HIV is not associated with the unusual malignancies such as B-cell lymphoma and Kaposi's sarcoma, commonly found in patients with AIDS. Shaw, G. M., et al., Science 226, 1165 (1984); Delli Bovi, P., et al., Cancer Research, 46, 6333 (1986); Groopman, J. E., et al., Blood 67, 612 (1986). Furthermore, HIV infected patients often show a wide variation in times of disease incubation and speed of disease progression. It is not known whether any specific infectious agent other than HIV can be responsible for the complex pathogenesis often seen in this disease. One such candidate, initially identified as a virus or virus-like infectious agent is M. fermentans (incognitus strain). See, Lo, S-C., et al., Am. J. Trop. Med. Hyg. 40, 213 (1989); Lo, S-C., et al., Am. J. Trop. Med. Hyg. 41, 601 (1989).
The recognition of the incognitus strain of M. fermentans as a possible causative or opportunistic agent associated with AIDS has instigated the search for other pathogenic mycoplasmas which may contribute to a better understanding, diagnosis, prognosis, and treatment of AIDS.